The overall objective of this proposal is to more clearly define the regulation of hemoglobin synthesis and erythropoiesis in normal and abnormal hematologic conditions. In particular, the role of the heme-regulated translational inhibitor (HRI) on protein synthesis in immature erythroid cells, especially reticulocytes, and the regulation of HRI by heme will be studied. HRI is a heme regulated protein kinase that phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF-2-alpha). The phosphorylation of the alpha subunit of eIF-2 substantially inhibits the recycling of translational initiation and thereby results in the cessation of protein synthesis. This proposal therefore seeks to clarify the physiologic function of HRI by studying the consequences of the ablation of this expression or in its activity both in cell culture and in transgenic animals. Three specific aims are listed: 1) to study the regulation of hemoglobin synthesis and late erythroid differentiation by HRI in murine erythroleukemia cells; 2) to study the role of HRI in the regulation of erythroid differentiation and HRI in the regulation of erythroid differentiation and hemoglobin synthesis in bone marrow stem cells in culture and in whole animals; and 3) to study the erythroid development and hemoglobin synthesis during embryonic development in adult mice by knock-out of the HRI gene.